The long-term objective of the research is to understand the mechanism of transcription, initiation and promoter site selection in eukaryotes. In order to achieve these objectives, the study of transcription of Adenovirus 2 (Ad 2) DNA in an in vitro system will be continued. The major components of the system under investigation are wheat germ, human placenta and calf thymus, class 11 polymerases and purified Ad 2 DNA. We have also used cloned promoter sequences of the Adenovirus genome to assess the fidelity of transcription in a completely purified system. Our research indicates that these polymerases can; (1) form strong binary complex with Ad 2 DNA, (2) these complexes formed are at specific sites on the DNA molecule which are similar or identical to in vivo promoters, (3) binary complexes exist in two different states; open, able to initiate in the presence of initiation inhibitors, and closed, which are inactivated by these inhibitors, (4) only a small fraction of the polymerase molecules can form binary complexes and transcribe double-stranded Ad 2 DNA, (5) transcription from these binary complexes constitutes ca. 70-80% of all transcription in our in vitro system, and (6) using a truncated template method, we showed that RNA synthesized from a template containing the major late promoter of Adenovirus was of the predicted size if the transcription started at the in vivo site and proceeded in the correct direction. The RNA of the expected size was the major product of this synthesis. The proposed research will now concentrate on answering the following questions: (1) What is the precise position of the binding site on DNA, (2) What are the sequences to which the two types of binary complexes are bound, (3) What is the relation between binding site and initiation site for these polymerases on different promoters, (4) What are the catalytic properties and the polypeptide composition of the polymerase which is capable of recognizing promoters and synthesizing RNA.